CN109324137B

CN109324137B - Method for determining content of rabdosia tenuiflora B in rabdosia tenuiflora medicinal material

Info

Publication number: CN109324137B
Application number: CN201811367805.4A
Authority: CN
Inventors: 杨雅欣; 邹娟; 徐文芬; 刘亚华; 周勋
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2021-11-30
Anticipated expiration: 2038-11-16
Also published as: CN109324137A

Abstract

The invention discloses a method for measuring the content of rabdosia lophanthide B in a rabdosia lophanthide medicinal material, which comprises the following steps: chromatographic conditions are as follows: thermo Accucore C18(4.6 mm. times.150 mm, 2.0 μm); mobile phase: the mobile phase A is methanol, the mobile phase B is 0.1 percent phosphoric acid, and isocratic elution is carried out; detection wavelength: 210-280 nm; column temperature: 20-30 ℃; flow rate: 0.8-1.2 mL/min^‑1(ii) a Sample introduction amount: 10 mu L of the solution; preparing a reference substance solution; preparing a test solution; and (4) measuring by an HPLC method. The content determination method of the rabdosia tenuiflora medicinal material is established by taking the rabdosia tenuiflora B as a determination index and adopting a high performance liquid chromatography, and an experimental basis is provided for quality control and evaluation research of the rabdosia tenuiflora medicinal material; the determination method is accurate, high in sensitivity, good in repeatability and reliable in result, and provides a basis for deep development and utilization of rabdosia tenuiflora medicinal materials and safe, effective and quality-controllable related preparations.

Description

Method for determining content of rabdosia tenuiflora B in rabdosia tenuiflora medicinal material

Technical Field

The invention relates to a method for measuring the content of rabdosia tenuiflora B in rabdosia tenuiflora medicinal materials, belonging to the technical field of medicines.

Background

Isodon coetsa (Buch. -ham. ex D. Don) Hara, a plant of the genus Rabdosia of the family Labiatae, is distributed in Guizhou, Yunnan, Tibet, Sichuan and Guangxi provinces in the southwest of China. It has good effect in treating hepatitis, atrophic gastritis and various tumors, and is widely applied in folk. Through literature reference and analysis, the quality control of rabdosia tapering is reported less, only the content of total flavone and volatile oil components in the rabdosia tapering is reported at present, and the research on the content determination of main active diterpene components is blank. In the experiment, the main active diterpene components in the rabdosia tenuiflora are separated and purified by various separation means such as column chromatography, liquid phase preparation and the like, and the diterpene compound rabdosia tenuiflora B with larger content is obtained through separation and purification by comparing nuclear magnetic data. According to the literature, the rabdosia tenuifolia B is the main active ingredient of the rabdosia tenuifolia medicinal material, and has obvious pharmacological effects of resisting tumors and cancers, treating leukemia and the like. The method is not beneficial to further development and utilization of the rabdosia tapering. In addition, the quality of the medicinal materials and related preparations is difficult to control without quantitative detection of the effective components, and the safety and effectiveness of clinical medication cannot be ensured.

Disclosure of Invention

The invention aims to provide a method for measuring the content of rabdosia lophanthide B in a rabdosia lophanthide medicinal material. The content determination method of the rabdosia tenuiflora medicinal material is established by taking the rabdosia tenuiflora B as a determination index and adopting a high performance liquid chromatography, and an experimental basis is provided for quality control and evaluation research of the rabdosia tenuiflora medicinal material; the determination method is accurate, high in sensitivity, good in repeatability and reliable in result, and provides a basis for deep development and utilization of rabdosia tenuiflora medicinal materials and safe, effective and quality-controllable related preparations.

In order to solve the technical problems, the invention adopts the following technical scheme: a method for measuring the content of rabdosia lophanthide B in a rabdosia lophanthide medicinal material comprises the following steps:

chromatographic conditions are as follows: thermo Accucore C₁₈4.6mm × 150mm, 2.0 μm; the mobile phase A is methanol, the mobile phase B is 0.1 percent phosphoric acid water solution, and isocratic elution is carried out; detection wavelength: 210-280 nm; column temperature: 20-30 ℃; flow rate: 0.8-1.2 mL/min^-1(ii) a Sample introduction amount: 10 mu L of the solution;

preparation of control solutions: precisely weighing herba Rabdosiae Glaucocalycis ethyl reference substance, placing in volumetric flask, adding 90-100% methanol solution to obtain 0.3000-0.6000 mg/mL solution^-1The control solution of (4);

preparation of a test solution: taking 1.8-2.2g of rabdosia tenuiflora medicinal material powder, precisely weighing, placing in a triangular conical flask, precisely adding 15-25 mL of 30-90% ethanol solution, weighing, performing reflux extraction, taking out, cooling, adding 30-90% ethanol solution to complement the loss weight, shaking uniformly, filtering, taking out the subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution;

and (3) determination by an HPLC method: respectively sucking the reference solution and the sample solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, measuring peak area value, and calculating content.

In the method for determining the content of the isodon acutangulata B in the isodon acutangulata medicinal material, the isocratic elution procedure is as follows: 0-20 min, 70% A.

In the method for measuring the content of the isodon acutangulata B in the isodon acutangulata medicinal material, the reflux extraction comprises the following steps: reflux extraction was carried out for 2h at a water bath temperature of 80 ℃.

In the method for measuring the content of the isodon excisoides in the isodon excisoides medicinal material, the detection wavelength is as follows: 234 nm; column temperature: 25 ℃; flow rate: 1 mL. min^-1。

In the method for measuring the content of the isodon acutangulata ethyl in the isodon acutangulata medicinal material, the preparation of the reference substance solution comprises the following steps: accurately weighing 12.50mg of Rabdosia trichocarpa reference substance, placing in a volumetric flask, adding methanol solution to prepare 0.5000 mg/mL^-1The control solution of (4).

In the method for measuring the content of rabdosia lophanthide B in the rabdosia lophanthide medicinal material, the preparation of the test solution comprises the following steps: taking 2.0g of rabdosia tenuiflora medicinal material powder, precisely weighing, placing the powder in a triangular conical flask, precisely adding 20mL of 65% ethanol solution, weighing, carrying out reflux extraction, taking out, cooling, adding 65% ethanol solution to supplement the loss weight, shaking up, filtering, taking out the subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution. The inventors conducted a number of experiments, and the following are partial experimental studies

Experimental example 1 examination of content measurement method

1 Instrument and reagent

1.1 instruments

Thermo UltiMate-3000 high performance liquid chromatograph (saimer, usa); a DAD detector; thermo Accucore C₁₈(4.6 mm. times.150 mm, 2.0 μm); an AG135 type electronic balance (Mettler-Toledo, switzerland); DK-98-II type water bath (Tester instruments, Inc., Tianjin) and the like.

1.2 reagents

Methanol (Tiandi corporation, chromatographic purity; Chemicals group, Inc., analytical purity, USA); ethanol (analytical purity, Fuyu fine chemical Co., Tianjin); phosphoric acid (Tianjin, Mimi European chemical reagent Co., Ltd., chromatographic purity); double distilled water; rabdosia lophanthide B (prepared in laboratories, purity 99.99%).

1.3 herbs

The test sample was collected by the inventor in Guizhou to open Yang and was accurately identified as Isodon coetsa (Buch. -ham. ex D.Don) Hara of Rabdosia in Labiatae by professor Zhao Junhua of Guiyang traditional Chinese medicine institute.

2 methods and results

2.1 chromatographic conditions

A chromatographic column: thermo Accucore C₁₈(4.6 mm. times.150 mm, 2.0 μm); mobile phase: methanol (a) was isocratically eluted with 0.1% phosphoric acid aqueous solution (B) (70% a); detection wavelength: 234 nm; column temperature: 25 ℃; flow rate: 1 mL. min^-1(ii) a Sample introduction amount: 10 μ L.

2.2 preparation of the solution

(1) Preparation of control solutions

Accurately weighing 12.50mg of Rabdosia trichocarpa ethyl reference substance, placing in a volumetric flask, adding methanol solution to prepare 0.5000 mg/mL^-1The control solution of (4).

(2) Preparation of test solution

Weighing about 2.0g of the powder, accurately weighing, placing in a 50mL conical flask, accurately adding 20mL of 65% ethanol solution, weighing, reflux-extracting (water bath temperature 80 deg.C) for 2h, taking out, cooling, adding 65% ethanol solution to make up for the lost weight, shaking, filtering, collecting the filtrate, and filtering with 0.45 μm microporous membrane to obtain the sample solution.

2.3 specialization examination

Precisely sucking 10 μ L of each of the reference solution, the sample solution and the blank solvent, and determining according to the established chromatographic conditions. The result shows that the retention time of a target peak to be detected in the chromatogram of the test solution and the chromatographic peak of the reference product of the rabdosia tenuiflora B are consistent with the UV spectrogram, the separation degree is more than 1.5, the baseline separation is achieved, the peak purity is more than 98 percent, and the established method has strong specificity. As shown in fig. 1.

2.4 inspection of Linear Range

Accurately sucking reference substance solution (0.5000 mg. mL) of Rabdosia trichocarpa ethyl^-1) Placing 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL into a 5mL volumetric flask, diluting with methanol solution to constant volume to scale, shaking up to prepare a series of concentration reference solutions, precisely sucking 10 μ L of the above solutions, and injecting into a high performance liquid chromatograph according to the proposed chromatographic conditions. And (3) drawing a standard curve by taking the sample amount (X) of the reference substance as an abscissa and the peak area value (Y) as an ordinate to obtain a regression equation Y which is 14.897X-0.2409 and r which is 0.9999, wherein the result shows that the sample amount of the rabdosia leptinoides B is in a good linear relation with the peak area value within the range of 1.0000-5.0000 mu g. As shown in table 1 and fig. 2.

Table 1 results of linear range investigation (n ═ 6)

2.5 precision test

Precisely sucking the reference solution (0.3000 mg. mL) of Rabdosia trichocarpa^-1) According to the drawn chromatographic conditions, sample introduction is continuously carried out for 6 times, 10 mu L of sample introduction is carried out each time, the peak area of the isodon excisoides component is measured (shown in figure 3), the RSD value of the calculated peak area is 0.08 percent, the technical requirement of quality standard analytical method verification (RSD is within 2.0 percent) is met, and the instrument precision is good. The results are shown in fig. 3 and table 2.

Table 2 precision test results (n ═ 6)

2.6 repeatability test

Taking the same batch of medicinal materials to be tested, preparing 6 parts of test solution according to a formulated test solution preparation method, respectively carrying out sample injection measurement according to formulated chromatographic conditions, wherein each time is 10 mu L, the average content of the Isodon lophanthoides is 3.46mg/g (shown in figure 4), calculating the RSD value of the content to be 0.90%, and meeting the technical requirement of quality standard analysis method verification (the RSD is within 3.0%), thereby indicating that the repeatability of the method is good. The results are shown in Table 3.

Table 3 results of repetitive tests (n ═ 6)

2.7 stability test

Taking the medicinal materials to be tested with the same batch number as the repeatability test, preparing 1 part of test solution according to a proposed method, injecting 10 μ L of sample at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h and 24h respectively under the proposed chromatographic conditions, and determining the peak area value of the Rabdosia trichocarpa component (see figure 5). The RSD value of the peak area is calculated to be 1.43 percent, and the result shows that the test solution is stable within 24 hours. The results are shown in Table 4.

Table 4 stability test results (n ═ 9)

2.8 sample recovery test

Taking 6 parts of medicinal materials to be detected in the same batch as the repeatability test, wherein each part is about 1.0g, precisely weighing, adding a reference substance of the isodon acutangulatum ethyl according to a ratio of 1:1, and respectively preparing the test solution according to a preparation method of the proposed test solution. According to proposed chromatographic conditions, respectively carrying out sample injection measurement, recording peak area values of chromatographic peaks of the rabdosia tenuiflora B (shown in figure 6), calculating that the average recovery rate of the rabdosia tenuiflora B is 94.89%, the RSD value is 1.10%, and meeting the technical requirements of quality standard analysis method verification (the limit of the recovery rate is 90-108%), thereby indicating that the accuracy of the measurement result of the method is high. The results are shown in Table 5.

TABLE 5 sample recovery test results (n ═ 6)

2.9 determination of the samples

Respectively taking 5 batches of the rabdosia tenuiflora medicinal material, wherein each batch is about 2.0g, precisely weighing, preparing a test solution according to a prepared test solution preparation method, respectively carrying out sample injection measurement according to prepared chromatographic conditions, repeatedly measuring each sample for 3 times, recording the peak area value of the rabdosia tenuiflora ethyl chromatographic peak (shown in figure 7), and calculating the content of the rabdosia tenuiflora ethyl in the dry medicinal material by using a standard curve method. The results are shown in Table 6.

TABLE 65 set of dried Rabdosia trichocarpa (n ═ 3)

Discussion of 3

3.1 chromatographic Condition selection

In the experiment, the 3D atlas of the DAD detector is adopted to screen the detection wavelength, and the result shows that the rabdosia gracilis B has the maximum absorption at the position of 234 nm. The method adopts solvent systems such as methanol-water, acetonitrile-0.1% phosphoric acid water and the like, results are not ideal, and methanol-0.1% phosphoric acid water is selected as a final mobile phase through further optimization. For ThermoAccucore C respectively₁₈(4.6 mm. times.150 mm, 2.0 μm), Elite Hypersil ODS (250 mm. times.4.6 mm, 5 μm), Agilent SB-C₁₈(150mm×4.6mm，5μm)，Pntulips BP-C₁₈The column (250mm multiplied by 4.6mm, 5 μm) and 4 different chromatographic columns were analyzed and compared, and the result showed that Thermo Accucore C₁₈(4.6mm×150mm，20 μm) column, high sensitivity, good peak shape and separation degree. The detection column temperatures of 20 ℃, 25 ℃ and 30 ℃ are respectively set, and the result shows that when the column temperature is 25 ℃, the retention time of the chromatographic peak of the Rabdosia trichocarpa is moderate, and the peak shape and the separation effect are good. The sample injection amounts of 5 muL, 10 muL, 15 muL and 20 muL are respectively set, and the result shows that the peak shape and the separation effect of the rabdosia tenuifolia B chromatographic peak are better when the sample injection amount is 10 muL.

3.2 examination of extraction conditions

35%, 50%, 65%, 80% and 95% ethanol solutions are respectively used as extraction solvents, and the results show that the 65% ethanol solution has the highest extraction rate, so that the ethanol solution is selected. The experiment compares the ultrasonic extraction method with the reflux extraction method, and the reflux extraction method is selected because the content of the rabdosia tapering bud ethyl element extracted by reflux is obviously superior to that extracted by ultrasonic extraction. The extraction time is set to be 1h, 1.5h, 2h, 2.5h and 3h respectively, and the result shows that the extraction rate of the rabdosia tenuiflora B is higher when the extraction time is 2h, so that the rabdosia tenuiflora B is selected. Setting 1: 8. 1: 10. 1: 12. 1: 14. 1: 16, and the result shows that the ratio of the materials to the liquid is 1: 10, the ratio of the material to the liquid is relatively high, so that the ratio of the material to the liquid is selected to be 1: 10. the determination method of the invention has the advantages of accuracy, high sensitivity, good repeatability and reliable result.

Compared with the prior art, the content determination method of the rabdosia tenuiflora medicinal material is established by taking the rabdosia tenuiflora B as a determination index and adopting a high performance liquid chromatography, and an experimental basis is provided for quality control and evaluation research of the rabdosia tenuiflora medicinal material; the determination method is accurate, high in sensitivity, good in repeatability and reliable in result, and provides a basis for deep development and utilization of rabdosia tenuiflora medicinal materials and safe, effective and quality-controllable related preparations.

Drawings

FIG. 1 is a diagram of a specific investigation HPLC chromatogram and UV spectrum (wherein A is a blank solution, B is a reference solution, C is a test solution, D is a reference solution UV spectrum, and E is a test solution UV spectrum);

FIG. 2 is a graph of the standard of Rabdosia trichocarpa ethyl;

FIG. 3 is a precision test HPLC chromatogram overlay;

FIG. 4 is a chromatogram overlay of a reproducibility test HPLC;

FIG. 5 is a stability test HPLC chromatogram overlay;

FIG. 6 is a sample recovery assay HPLC chromatogram overlay;

FIG. 7 is a HPLC chromatogram overlay of 5 batches of Isodon conoides medicinal material samples.

The present invention will be further described with reference to the following examples.

Detailed Description

Example 1:

a method for measuring the content of rabdosia lophanthide B in a rabdosia lophanthide medicinal material comprises the following steps:

chromatographic conditions Thermo Accucore C₁₈(4.6 mm. times.150 mm, 2.0 μm); the mobile phase A is methanol, the mobile phase B is 0.1 percent phosphoric acid water solution, and isocratic elution is carried out; the elution procedure was: 0-20 min, 70% A;

detection wavelength: 234 nm; column temperature: 25 ℃; flow rate: 1 mL. min^-1(ii) a Sample introduction amount: 10 μ L.

Preparation of control solutions: precisely weighing herba Coriandri B reference substance, placing in a volumetric flask, adding 95% methanol solution to obtain 0.4500 mg/mL solution^-1The control solution of (4).

Preparation of a test solution: taking 2.0g of rabdosia tenuiflora medicinal material powder, precisely weighing, placing the powder in a triangular conical flask, precisely adding 20mL of 65% ethanol solution, weighing, performing reflux extraction, taking out, cooling, adding 65% ethanol solution to supplement the loss weight, shaking up, filtering, taking a subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a sample solution.

And (3) determination by an HPLC method: respectively sucking the reference solution and the sample solution, injecting into a liquid chromatograph, measuring according to the above chromatographic conditions, measuring peak area value, and calculating content by standard curve method.

Claims

1. A method for measuring the content of rabdosia tenuiflora B in rabdosia tenuiflora medicinal materials is characterized by comprising the following steps: the method for measuring the content of the rabdosia lophanthide B comprises the following steps:

chromatographic conditions are as follows: thermo Accucore C₁₈4.6mm × 150mm, 2.0 μm; the mobile phase A is methanol, the mobile phase B is 0.1 percent phosphoric acid water solution, and isocratic elution is carried out; detection wavelength: 234 nm; column temperature: 25 ℃; flow rate: 1 mL. min^-1(ii) a Sample introduction amount: 10 mu L of the solution; the isocratic elution procedure was: 0-20 min, 70% A;

preparation of a test solution: taking 1.8-2.2g of rabdosia tenuiflora medicinal material powder, precisely weighing, placing in a triangular conical flask, precisely adding 15-25 mL of 30-90% ethanol solution, weighing, performing reflux extraction, taking out, cooling, adding 30-90% ethanol solution to complement the loss weight, shaking uniformly, filtering, taking out the subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution; the reflux extraction comprises the following steps: reflux-extracting at 80 deg.C in water bath for 2 hr;

and (3) determination by an HPLC method: respectively sucking the reference solution and the test solution, injecting into a liquid chromatograph, measuring according to the chromatographic conditions, measuring peak area value and calculating content;

the control solution was prepared by: accurately weighing 12.50mg of Rabdosia trichocarpa reference substance, placing in a volumetric flask, adding methanol solution to prepare 0.5000 mg/mL^-1The control solution of (4);

the preparation of the test solution is as follows: taking 2.0g of rabdosia tenuiflora medicinal material powder, precisely weighing, placing the powder in a triangular conical flask, precisely adding 20mL of 65% ethanol solution, weighing, carrying out reflux extraction, taking out, cooling, adding 65% ethanol solution to supplement the loss weight, shaking up, filtering, taking out the subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution.